RESUMEN
Influenza A viruses cause lung disease via an incompletely understood mechanism that involves the accumulation of liquid within the lungs. The accumulation of lung liquid is normally prevented by epithelial Na(+) absorption, a transport process regulated via several pathways including phosphoinositide-3-kinase (PI3K). Since the influenza A virus encodes a non-structural protein (NS1) that can activate this kinase, we now explore the effects of NS1 upon the biophysical properties of human airway epithelial cells. Transient expression of NS1 depolarized electrically isolated cells maintained in glucocorticoid-free medium by activating a cation conductance identical to the glucocorticoid-induced conductance seen in single cells. This response involved PI3K-independent and PI3K-dependent mechanisms. Infecting glucocorticoid-deprived cells with influenza A virus disrupted the normal electrical coupling between neighbouring cells, but also activated a conductance identical to that induced by NS1. This response to virus infection was only partially dependent upon NS1-mediated activation of PI3K. The presence of NS1 allows influenza A to modify the biophysical properties of infected cells by activating a Na(+)-permeable conductance. Whilst the activation of Na(+)-permeable channels may be expected to increase the rate of Na(+) absorption and thus reduce the volume of liquid in the lung, liquid does normally accumulate in influenza A-infected lungs. The overall effect of influenza A on lung liquid volume may therefore reflect a balance between the activation and inhibition of Na(+)-permeable channels.
Asunto(s)
Virus de la Influenza A/patogenicidad , Canales Iónicos/metabolismo , Sistema Respiratorio/metabolismo , Sistema Respiratorio/virología , Fenómenos Biofísicos , Línea Celular , Dexametasona/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Virus de la Influenza A/genética , Gripe Humana/metabolismo , Gripe Humana/virología , Transporte Iónico/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Sistema Respiratorio/citología , Sodio/metabolismo , Transfección , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/toxicidadRESUMEN
In the fetus, there is a net secretion of liquid (LL) by the lung as a result of active transport of chloride ions. The rate of secretion and the resulting volume of LL are vital for normal lung growth but how volume is sensed and how secretion may be regulated are still unknown. Towards term under the influence of thyroid and adrenocorticoid hormones, the epithelial sodium channel (ENaC) is increasingly expressed in the pulmonary epithelium. Adrenaline released by the fetus during labour activates ENaC and produces rapid absorption of liquid in preparation for air breathing; absence of ENaC is incompatible with survival. There may be other mechanisms involved in aiding liquid clearance including changes in epithelial permeability, an effect of oxygen on both ENaC and Na/K ATPase and perhaps the influence of additional hormones on ENaC activity. Some time after birth there are further developmental changes with the appearance of other cation channels (CNG1 and perhaps NSCC) which contribute to the liquid absorptive side of the balance existing across the epithelium between secretion and absorption to produce essentially almost no net liquid movement in the postnatal lung. The evidence for these processes is discussed and areas of uncertainty indicated.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Madurez de los Órganos Fetales/fisiología , Pulmón/embriología , Mucosa Respiratoria/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Compartimentos de Líquidos Corporales , Humanos , Transporte Iónico , Pulmón/metabolismo , Mucosa Respiratoria/embriologíaRESUMEN
Academic paediatrics is an exciting and rewarding career path but is not immune to the problems of recruitment and retention currently affecting most branches of medicine. The Modernising Medical Careers initiative, with its explicit academic training path, offers an unparalleled opportunity to develop novel schemes that promote recruitment and retention. Coordinated action is required to define, publicise and support the new academic training programmes and to attract the best trainees into them.
Asunto(s)
Selección de Profesión , Pediatría/educación , Educación de Postgrado en Medicina/organización & administración , Docentes Médicos , Becas , Relaciones Interprofesionales , Satisfacción en el Trabajo , Selección de Personal , Reino Unido , Recursos HumanosRESUMEN
BACKGROUND AND PURPOSE: Absorptive epithelia express apical receptors that allow nucleotides to inhibit Na(+) transport but ATP unexpectedly stimulated this process in an absorptive cell line derived from human bronchiolar epithelium (H441 cells) whilst UTP consistently caused inhibition. We have therefore examined the pharmacological basis of this anomalous effect of ATP. EXPERIMENTAL APPROACH: H441 cells were grown on membranes and the short circuit current (I(SC)) measured in Ussing chambers. In some experiments, [Ca(2+)](i) was measured fluorimetrically using Fura -2. mRNAs for adenosine receptors were determined by the polymerase chain reaction (PCR). KEY RESULTS: Cross desensitization experiments showed that the inhibitory response to UTP was abolished by prior exposure to ATP whilst the stimulatory response to ATP persisted in UTP-pre-stimulated cells. Apical adenosine evoked an increase in I(SC) and this response resembled the stimulatory component of the response to ATP, and could be mimicked by adenosine receptor agonists. Pre-stimulation with adenosine abolished the stimulatory component of the response to ATP. mRNA encoding A(1), A(2A) and A(2B) receptor subtypes, but not the A(3) subtype, was detected in H441 cells and adenosine receptor antagonists could abolish the ATP-evoked stimulation of Na(+) absorption. CONCLUSIONS AND IMPLICATIONS: The ATP-induced stimulation of Na(+) absorption seems to be mediated via A(2A/B) receptors activated by adenosine produced from the extracellular hydrolysis of ATP. The present data thus provide the first description of adenosine-evoked Na(+) transport in airway epithelial cells and reveal a previously undocumented aspect of the control of this physiologically important ion transport process.
Asunto(s)
Adenosina/farmacología , Mucosa Respiratoria/metabolismo , Sodio/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Transporte Biológico Activo/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Interpretación Estadística de Datos , Colorantes Fluorescentes , Fura-2 , Humanos , Antagonistas de Receptores Purinérgicos P1 , ARN/biosíntesis , ARN/genética , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y2 , Mucosa Respiratoria/citología , Mucosa Respiratoria/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Electrophysiological studies of H441 human distal airway epithelial cells showed that thapsigargin caused a Ca(2+)-dependent increase in membrane conductance (G(Tot)) and hyperpolarization of membrane potential (V(m)). These effects reflected a rapid rise in cellular K(+) conductance (G(K)) and a slow fall in amiloride-sensitive Na(+) conductance (G(Na)). The increase in G(Tot) was antagonized by Ba(2+), a nonselective K(+) channel blocker, and abolished by clotrimazole, a KCNN4 inhibitor, but unaffected by other selective K(+) channel blockers. Moreover, 1-ethyl-2-benzimidazolinone (1-EBIO), which is known to activate KCNN4, increased G(K) with no effect on G(Na). RT-PCR-based analyses confirmed expression of mRNA encoding KCNN4 and suggested that two related K(+) channels (KCNN1 and KCNMA1) were absent. Subsequent studies showed that 1-EBIO stimulates Na(+) transport in polarized monolayers without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)), suggesting that the activity of KCNN4 might influence the rate of Na(+) absorption by contributing to G(K). Transient expression of KCNN4 cloned from H441 cells conferred a Ca(2+)- and 1-EBIO-sensitive K(+) conductance on Chinese hamster ovary cells, but this channel was inactive when [Ca(2+)](i) was <0.2 microM. Subsequent studies of amiloride-treated H441 cells showed that clotrimazole had no effect on V(m) despite clear depolarizations in response to increased extracellular K(+) concentration ([K(+)](o)). These findings thus indicate that KCNN4 does not contribute to V(m) in unstimulated cells. The present data thus establish that H441 cells express KCNN4 and highlight the importance of G(K) to the control of Na(+) absorption, but, because KCNN4 is quiescent in resting cells, this channel cannot contribute to resting G(K) or influence basal Na(+) absorption.
Asunto(s)
Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Mucosa Respiratoria/fisiología , Animales , Bencimidazoles/farmacología , Células CHO , Agonistas de los Canales de Calcio/farmacología , Línea Celular , Linaje de la Célula , Clonación Molecular , Cricetinae , Inhibidores Enzimáticos/farmacología , Expresión Génica/fisiología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potasio/metabolismo , ARN Mensajero/metabolismo , Mucosa Respiratoria/citología , Sodio/metabolismo , Tapsigargina/farmacologíaRESUMEN
Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na(+) channel alpha- and beta-subunits and increased transepithelial ion transport (measured as short-circuit current, I(sc)) from <4 microA.cm(-2) to 10-20 microA.cm(-2). This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil >or= amiloride > EIPA and can thus be attributed to active Na(+) absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na(+) pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na(+) conductance (G(Na)) from a negligible value to 100-200 microS.cm(-2). Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na(+) and Li(+) and that the permeability to these cations was approximately fourfold greater than to K(+). There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an approximately 60% increase in I(sc), and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in G(Na). This cAMP-dependent control over G(Na) was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na(+) transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Canales de Sodio/metabolismo , Membrana Celular/metabolismo , Polaridad Celular/fisiología , Células Cultivadas , Colforsina/farmacología , Canales Epiteliales de Sodio , Humanos , Potenciales de la Membrana/efectos de los fármacos , ARN Mensajero/análisis , Mucosa Respiratoria/citología , Sodio/metabolismo , Canales de Sodio/genéticaRESUMEN
Secretion of HCO(3)(-) by airway submucosal glands is essential for normal liquid and mucus secretion. Because the liquid bathing the airway surface (ASL) is acidic, it has been proposed that the surface epithelium may acidify HCO(3)(-)-rich glandular fluid. The aim of this study was to investigate the mechanisms by which intact distal bronchi, which contain both surface and glandular epithelium, modify pH of luminal fluid. Distal bronchi were isolated from pig lungs, cannulated in a bath containing HCO(3)(-)-buffered solution, and perfused continually with an aliquot of similar, lightly buffered solution (LBS) in which NaCl replaced NaHCO(3)(-) (pH 7 with NaOH). The pH of this circulating LBS initially acidified (by 0.053 +/- 0.0053 pH units) and transepithelial potential difference (PD) depolarized. The magnitude of acidification was increased when pH(LBS) was higher. This acidification was unaffected by luminal dimethylamiloride (DMA, 100 microM) but was inhibited by 100 nM bafilomycin A(1) (by 76 +/- 13%), suggesting involvement of vacuolar-H(+) ATPase. Addition of ACh (10 microM) evoked alkalinization of luminal LBS and hyperpolarization of transepithelial PD. The alkalinization was inhibited in HCO(3)(-)-free solutions containing acetazolamide (1 mM) and by DMA and was enhanced by bumetanide (100 microM), an inhibitor of Cl(-) secretion. The hyperpolarization was unaffected by these maneuvers. The anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (300 microM) and combined treatment with DMA and bumetanide blocked both the alkalinization and hyperpolarization responses to ACh. These results are consistent with earlier studies showing that ACh evokes glandular secretion of HCO(3)(-) and Cl(-). Isolated distal airways thus secrete both acid and base equivalents.
Asunto(s)
Equilibrio Ácido-Base/fisiología , Amilorida/análogos & derivados , Bronquios/metabolismo , Macrólidos , Bicarbonato de Sodio/metabolismo , Acetilcolina/farmacología , Equilibrio Ácido-Base/efectos de los fármacos , Ácidos/metabolismo , Álcalis/metabolismo , Amilorida/farmacología , Animales , Antibacterianos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Tampones (Química) , Cloruros/metabolismo , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , PorcinosRESUMEN
Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (approximately 48 h) on permeable supports in medium devoid of hormones and growth factors whilst P(O2) was maintained at the level found in either the fetal (23 mmHg) or the postnatal (100 mmHg) alveolar regions. The cells became incorporated into epithelial layers that generated a basal short-circuit current (I(SC)) attributable to spontaneous Na(+) absorption. Cells at neonatal P(O2) generated larger currents than did cells at fetal P(O2), indicating that this Na(+) transport process is oxygen sensitive. Irrespective of P(O2), isoprenaline failed to elicit a discernible change in I(SC), demonstrating that beta-adrenoceptor agonists do not stimulate Na(+) transport under these conditions. However, isoprenaline did elicit cAMP accumulation in these cells, indicating that functionally coupled beta-adrenoceptors are present. Further experiments showed that isoprenaline did increase I(SC) in cells treated (24 h) with a combination of tri-iodothyronine (T(3), 10 nM) and dexamethasone (200 nM). Studies of basolaterally permeabilised cells showed that these hormones are essential for the isoprenaline-evoked increase in the apical membrane's Na(+) conductance (G(Na)), whereas isoprenaline-evoked changes in apical Cl(-) conductance (G(Cl)) can occur in both control and hormone-treated cells. Irrespective of their hormonal status, FDLE cells thus express beta-adrenoceptors that are functionally coupled to adenylate cyclase, and allow beta-adrenoceptor agonists to modulate the apical membrane's anion conductance. However, T(3) and dexamethasone are needed if these receptors are to exert control over G(Na). These hormones may thus play an important role in the functional maturation of the lung by allowing beta-adrenoceptor-mediated control over epithelial Na(+) channels in the apical plasma membrane.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Pulmón/embriología , Triyodotironina/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Cloruros/fisiología , Medios de Cultivo , AMP Cíclico/biosíntesis , Combinación de Medicamentos , Conductividad Eléctrica , Epitelio/embriología , Feto/citología , Feto/efectos de los fármacos , Feto/metabolismo , Isoproterenol/farmacología , Oxígeno/metabolismo , Presión Parcial , Ratas , Sodio/fisiologíaRESUMEN
Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current (I(sc)) that was inhibited (~80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na(+) from the apical solution. Isoprenaline increased the amiloride-sensitive I(sc) about twofold. Experiments in which apical membrane Na(+) currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na(+) conductance (G(Na(+))). Isoprenaline also increased apical Cl- conductance (G(Cl-)) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The isoprenaline-evoked changes in G(Na(+)) and G(Cl(minus sign)) could account for the changes in I(sc) observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of I(sc) or G(Na(+)) demonstrating that the rise in G(Cl-) is not essential to the stimulation of Na(+) transport.
Asunto(s)
Alveolos Pulmonares/metabolismo , Receptores Adrenérgicos beta/metabolismo , Mucosa Respiratoria/metabolismo , Sodio/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Bario/farmacología , Polaridad Celular/fisiología , Células Cultivadas , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Cámaras de Difusión de Cultivos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Agua Pulmonar Extravascular/metabolismo , Femenino , Feto/citología , Gliburida/farmacología , Hipoglucemiantes/farmacología , Isoproterenol/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Embarazo , Alveolos Pulmonares/citología , Ratas , Mucosa Respiratoria/citología , Canales de Sodio/metabolismoRESUMEN
The Calu-3 human cell line exhibits features of submucosal gland serous cells and secretes HCO(3)(-). The aim of this study was to identify the HCO(3)(-) transporters present in these cells by studying their role in the regulation of intracellular pH (pH(i)). Calu-3 cells were grown on coverslips, loaded with the pH-sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pH(i). Cells were acidified with NH(4)Cl (25 mM, 1 min) and pH(i) recovery recorded. In the absence of HCO(3)(-), initial recovery was 0.208 +/- 0.016 pH units min(-1) (n = 37). This was almost abolished by removal of extracellular Na(+) and by amiloride (1 mM), consistent with the activity of a Na(+)-H(+) exchanger (NHE). In the presence of HCO(3)(-) and CO(2), recovery (0.156 +/- 0.018 pH units min(-1)) was abolished (reduced by 91.8 +/- 6.7 %, n = 7) by removal of Na(+) but only attenuated (by 63.3 +/- 5.8 %, n = 9) by amiloride. 4,4-Dinitrostilbene-2,2-disulfonic acid (DNDS) inhibited recovery by 45.8 +/- 5.0 % (n = 7). The amiloride-insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K(+)] in the presence of either valinomycin or the K(+) channel opener 1-EBIO. In addition, forskolin (10 microM), which activates the cystic fibrosis transmembrane conductance regulator Cl(-) conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl(-) trebled pH(i) recovery rates, suggesting that an electroneutral, DNDS-sensitive, Cl(-)-HCO(3)(-) exchanger together with a NHE may be involved in pH(i) regulation and HCO(3)(-) secretion in these cells. RT-PCR detected the expression of the electrogenic Na(+)-HCO(3)(-) cotransporter NBC1 and the Cl(-)-HCO(3)(-) exchanger (AE2) but not the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1.
Asunto(s)
Hidrógeno/metabolismo , Membranas Intracelulares/metabolismo , Sistema Respiratorio/metabolismo , Membrana Serosa/metabolismo , Amilorida/farmacología , Bicarbonatos/farmacología , Tampones (Química) , Dióxido de Carbono/farmacología , Línea Celular , Membrana Celular/fisiología , Colforsina/farmacología , HEPES/farmacología , Homeostasis , Humanos , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/fisiología , Sistema Respiratorio/citología , Sistema Respiratorio/efectos de los fármacos , Membrana Serosa/citología , Membrana Serosa/efectos de los fármacos , Sodio/fisiología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/fisiología , Soluciones , Estilbenos/farmacologíaRESUMEN
Imposing hypoxia (P(O(2)) = 23 mmHg) upon A549 cells elicited increased G(amil) although previous work had predicted a fall in this parameter. G(amil) appeared to be dependent upon glucocorticoid-driven gene expression, a process inhibited by ERK, an enzyme activated by oxidative stress. However, hypoxia transiently activated this enzyme and the response was blocked by glucocorticoids, showing that the rise in G(amil) occurs only if ERK activation is suppressed. Fluorimetric assays showed that lowering P(O(2)) elicited H(2)O(2) formation indicating that this maneuver actually imposes oxidative stress, thus explaining how hypoxia can elicit responses normally associated with a rise in P(O(2)).
Asunto(s)
Amilorida/farmacología , Alveolos Pulmonares/citología , Mucosa Respiratoria/fisiología , Sodio/metabolismo , Animales , Hipoxia de la Célula , Línea Celular , Dexametasona/farmacología , Conductividad Eléctrica , Glucocorticoides/farmacología , Antagonistas de Hormonas/farmacología , Peróxido de Hidrógeno/metabolismo , Transporte Iónico , Mifepristona/farmacología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo , Oxígeno/fisiología , Presión , Mucosa Respiratoria/efectos de los fármacosRESUMEN
Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2 (100 mmHg) than at fetal PO2 (23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2 equivalent to that seen at birth, no rise in ISC was seen after 6 h but the response was fully established by 24 h. Studies of basolaterally permeabilised cells revealed a small rise in apical Na+ conductance (GNa) 6 h after PO2 was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h. Reporter gene assays showed that no activation of the -ENaC (epithelial Na+ channel -subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. Studies of apically permeabilised cells showed that a small rise in Na+ pump capacity was evident 6 h after PO2 was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISC thus develops 6-24 h after PO2 is raised and is due, primarily, to increased Na+ pump capacity. The increase in GNa thus coincides with activation of the -ENaC promoter but these effects occur after the rise in ISC is fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+ transport and not its cause.
Asunto(s)
Células Epiteliales/metabolismo , Pulmón/metabolismo , Oxígeno/metabolismo , Canales de Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Amilorida/farmacología , Animales , Polaridad Celular , Células Cultivadas , Genes Reporteros , Transporte Iónico , Pulmón/citología , Pulmón/embriología , Potenciales de la Membrana/fisiología , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Subunidades de Proteína , Ratas , Ratas Sprague-Dawley , Canales de Sodio/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Factores de Tiempo , Transcripción GenéticaRESUMEN
Electrophoretic mobility shift assays revealed minimal levels of NF-kappaB activity in rat distal lung epithelial cells cultured at fetal (23 mmHg) or adult alveolar (100 mmHg) P(O2), but revealed significant activation of this transcription factor in cells exposed to a rise in P(O2) mimicking that experienced at birth. This response was entirely abolished by pretreating cells with 5 mM sulfasalazine (SSA). This shift in P(O2) also evoked a rise in apical Na+ conductance (G(Na+)) that may underlie the O2-evoked stimulation of Na+ transport seen in these cells. Pretreatment with SSA had no effect upon G(Na+) in cells cultured continually at adult or fetal P(O2) but did inhibit the increase in G(Na+) seen in cells that had experienced the rise in P(O2). O2-evoked activation of NF-kappaB may thus mediate the increased Na+ transport that occurs when the distal lung epithelial cells are exposed to a physiologically-relevant increase in P(O2).
Asunto(s)
FN-kappa B/metabolismo , Oxígeno/farmacología , Alveolos Pulmonares/fisiología , Sodio/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Feto , Potenciales de la Membrana/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Embarazo , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Ratas , Canales de Sodio/fisiología , Sulfasalazina/farmacologíaRESUMEN
The O(2) and redox-sensitive transcription factors hypoxia inducible factor-1alpha (HIF-1alpha) and nuclear factor-kappaB (NF-kappaB) are differentially regulated in the alveolar epithelium over fetal to neonatal oxygen tensions. We have used fetal alveolar type II epithelial cells to monitor their regulation in association with redox responsiveness to antioxidant pretreatment in vitro. N-Acetyl-l-cysteine, a glutathione (GSH) precursor and a potent scavenger of reactive oxygen species, induced HIF-1alpha and ameliorated NF-kappaB nuclear abundance and DNA binding activity, respectively, in a dose-dependent manner. Analysis of variations in glutathione homeostasis at ascending DeltapO(2) regimen with N-acetyl-(L)-cysteine reveals increased GSH at the expense of the oxidized form of glutathione (GSSG), thereby shifting GSH/GSSG into reduction equilibrium. Pyrrolidine dithiocarbamate (PDTC), which exerts both antioxidant and pro-oxidant effects, provoked a substantial increase in HIF-1alpha nuclear abundance, with no apparent effect on its activation. PDTC reduced NF-kappaB nuclear abundance and its inhibitory effects on binding activity are dose-dependent. Assessment of glutathione homeostasis with PDTC shows increasing levels of GSSG at the expense of GSH, lowering GSH/GSSG in favor of an oxidative equilibrium. Our results indicate the hypoxic activation of HIF-1alpha and the hyperoxic induction of NF-kappaB in the fetal epithelium is redox-sensitive and, thus, tightly regulated by the GSH/GSSG equilibrium. This highlights glutathione as a key regulatory component for determining genetic responsiveness to oxidant/antioxidant imbalance in normal lung development and pathophysiological conditions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Disulfuro de Glutatión/metabolismo , Glutatión/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Alveolos Pulmonares/metabolismo , Mucosa Respiratoria/metabolismo , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Núcleo Celular/metabolismo , Células Cultivadas , Feto , Regulación de la Expresión Génica , Homeostasis , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Cinética , Oxidantes/farmacología , Oxidación-Reducción , Pirrolidinas/farmacología , Ratas , Tiocarbamatos/farmacología , Factores de TranscripciónRESUMEN
Isolated segments of porcine tracheal epithelium were mounted in Ussing chambers, current required to maintain transepithelial potential difference at 0 mV (short circuit current, I(SC)) was monitored and effects of nucleotides upon I(SC) were studied. Mucosal UTP (100 microM) evoked a transient rise in I(SC) that was followed by a sustained fall below basal I(SC) maintained for 30 min. Mucosal ATP (100 microM) also stimulated a transient rise in I(SC) but in contrast to UTP did not inhibit basal I(SC). Submucosal UTP and ATP both transiently increased I(SC). UTP-prestimulated epithelia were refractory to ATP but prestimulation with ATP did not abolish the response to UTP. The epithelia thus appear to express two populations of apical receptors allowing nucleotides to modulate I(SC). The UTP-induced rise was reduced by pretreatment with either bumetanide (100 microM), diphenylamin-2-carboxylic acid (DPC, 1 mM), or Cl(-) and HCO(3)(-)-free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 microM) abolished the UTP-induced increase in I(SC) but not the subsequent decrease. Staurosporine (0.1 microM) inhibited basal I(SC) and blocked UTP-induced inhibition of I(SC). Inhibitors of either protein kinase C (PKC) (D-erythro sphingosine) or PKA (H89) had no effect. This study suggests that UTP stimulates Cl(-) secretion and inhibits basal Na(+) absorption. ATP has a similar stimulatory effect, which may be mediated by activation of P2Y(2) receptors and an increase in [Ca(2+)](in), but no inhibitory effect, which is likely mediated by activation of a pyrimidine receptor and possible inhibition of a protein kinase other than PKC or PKA.
Asunto(s)
Adenosina Trifosfato/farmacología , Péptidos y Proteínas de Señalización Intracelular , Transporte Iónico/efectos de los fármacos , Tráquea/efectos de los fármacos , Uridina Trifosfato/farmacología , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Portadoras/farmacología , Interacciones Farmacológicas , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Técnicas In Vitro , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Porcinos , Tapsigargina/farmacología , Tráquea/citología , Tráquea/metabolismoRESUMEN
1. Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (for 48 h) at PO2 levels between 23 and 142 mmHg. Higher PO2 levels between 23 and 142 mmHg. Higher PO2 was associated with increased short circuit current (ISC) and increased abundance of the Na+ channel protein alpha-ENaC. PO2 had no effect upon ISC remaining after apical application of amiloride (10 microM). 2. Studies of cells maintained (for 48 h) at PO2 levels of 23 mmHg or 100 mmHg, and subsequently nystatin permeabilized (50 microM), showed that high PO2 increased Na+ pump capacity. This response was apparent 24 h after PO2 was raised whilst it took 48 h for the rise in ISC seen in intact cells to become fully established. Both parameters were unaffected by raising PO2 for only 30 min. 3. Basolateral application of isoprenaline (10 microM) did not affect ISC in cells maintained at 23 mmHg but evoked progressively larger responses at higher PO2. The response seen at 142 mmHg was larger than at 100 mmHg, the normal physiological alveolar PO2. 4. Isoprenaline had no effect on Na+ pump capacity at PO2 levels of 23 mmHg or 100 mmHg, but stimulated Na+ extrusion at 142 mmHg. Increasing PO2 above normal physiological levels thus allows the Na+ pump to be controlled by isoprenaline. This may explain the enhanced sensitivity to isoprenaline seen under these slightly hyperoxic conditions. 5. Changes in PO2 mimicking those occurring at birth thus exert profound influence over Na+ transport in FDLE cells and the Na+ pump could be an important locus at which this control is exercised.
Asunto(s)
Pulmón/metabolismo , Oxígeno/fisiología , Agonistas Adrenérgicos beta/farmacología , Amilorida/farmacología , Animales , Cardiotónicos/farmacología , Células Cultivadas , Diuréticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Isoproterenol/farmacología , Pulmón/citología , Pulmón/embriología , Ouabaína/farmacología , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Ratas , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacosRESUMEN
We have studied factors that potentially modulate the expression of mRNA coding for subunits of the amiloride-sensitive sodium channel, alphaENaC and betaENaC, in lungs of vaginally and Caesarean (CS)-delivered late gestation fetal guinea-pigs. Expression of alphaENaC and betaENaC mRNAs was developmentally regulated in the late gestation fetus, reaching peak levels at term (68 days post conception, PC) and postnatally, respectively. In animals delivered by CS at 65 days PC and term, alphaENaC mRNA expression was significantly increased by day 1 post partum, reaching levels greater than those normally achieved in vaginally delivered animals at term. In contrast, betaENaC mRNA levels remained significantly lower postnatally in animals delivered by CS at 65 days PC compared with those in vaginally and CS-delivered animals at term. Plasma cortisol and total triiodothyronine (T3) levels increased towards term, were higher 1 day after vaginal delivery but declined towards pre-term levels by day 3. Cortisol levels also increased rapidly in the CS-delivered animals, reaching levels similar to those in vaginally delivered animals at day 1. Plasma T3 levels at days 1 and 3 were significantly lower in animals delivered by CS at 65 days PC. The increase in alphaENaC mRNA paralleled the increase in plasma cortisol after delivery, but not T3, and inhibition of cortisol synthesis with 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) after CS delivery suppressed the increase in alphaENaC mRNA expression. Concomitant with the increase in alphaENaC mRNA expression after CS delivery at 65 days PC was an increase in the amiloride-blockable component of lung fluid clearance by day 3 postnatally. We conclude that in late gestation guinea-pigs delivered by CS there is a significant increase in lung alphaENaC expression postnatally, which is mediated, in part, by the postnatal rise in cortisol at delivery. This in turn leads to an increase in amiloride-sensitive lung fluid clearance, which is unrelated to labour.
Asunto(s)
Parto Obstétrico/métodos , Hormonas/sangre , Pulmón/metabolismo , Canales de Sodio/metabolismo , Animales , Animales Recién Nacidos , Líquidos Corporales/metabolismo , Cesárea , Ambiente , Canales Epiteliales de Sodio , Feto , Edad Gestacional , Cobayas , Hidrocortisona/biosíntesis , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Metirapona/farmacología , Oxígeno , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Respiración , Canales de Sodio/genéticaRESUMEN
P2Y receptor agonists stimulate Cl- secretion across both normal and cystic fibrosis (CF) airway epithelia, and therefore have potential for use in the treatment of CF. Although CF pathology is manifest primarily in the distal airways, most studies of P2Y-receptor-mediated airway epithelial ion transport have used cells cultured from proximal regions. Here we report the results of studies of P2Y-receptor-mediated ion transport in distal bronchi isolated from porcine lungs, cannulated and perfused. A luminal microelectrode was used to record transepithelial potential difference (PD) and cable analysis was applied to determine resistance (Rt) and equivalent short-circuit current (I(SC)). Luminal UTP (100 micromol/l) transiently hyperpolarized PD (from -5.8+/-0.3 to -6.5+/-0.4 mV) and increased I(SC) (from 47+/-6 to 55+/-8 microA cm(-2)) before inhibiting PD to below the pre-UTP level (-5.0+/-0.4 mV). The decline was attenuated by pretreatment with amiloride, and additional treatment with bumetanide inhibited the initial hyperpolarization, suggesting that UTP stimulates Cl- secretion and inhibits Na+ absorption across distal bronchi. Luminal addition of P2Y1 [ADP, 2-methylthio-ATP (2MeSATP)] and P2Y6 (UDP) receptor agonists had no effect on ion transport. Pretreatment with thapsigargin (0.3 micromol/l) did not prevent the UTP-induced increase in PD and I(SC) but attenuated the secondary fall in PD. Pretreatment with BAPTA/AM (50 micromol/l), however, had no effect on the response to UTP. Additional studies of isolated bronchial epithelial cells using Fura-2 showed that UTP increases [Ca2+]in, and this increase is blocked by pretreatment with thapsigargin. These results suggest that in intact distal bronchi luminal UTP stimulates Cl- secretion by a Ca2+-independent mechanism and inhibits Na+ absorption by a Ca2+-dependent mechanism. Both effects are likely to favour increased hydration of the airway surface, and may therefore be beneficial in CF.
Asunto(s)
Bronquios/metabolismo , Cloruros/metabolismo , Nucleótidos/farmacología , Receptores Purinérgicos P2/fisiología , Sodio/metabolismo , Absorción , Adenosina Difosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Conductividad Eléctrica , Microelectrodos , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y2 , Porcinos , Tapsigargina/farmacología , Tionucleótidos/farmacología , Uridina Difosfato/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
1 Rat foetal distal lung epithelial cells were plated onto permeable supports where they became integrated into epithelial sheets that spontaneously generated short circuit current (ISC). 2 Apical ATP (100 microM) evoked a transient fall in ISC that was followed by a rise to a clear peak which, in turn, was succeeded by a slowly developing decline to a value below control. Apical UTP evoked an essentially identical response. 3 UDP and ADP were ineffective whilst ATP had no effect when added to the basolateral solution. These effects thus appear to be mediated by apical P2Y2 receptors. 4 The rising phase of the responses to ATP/UTP was selectively inhibited by anion transport inhibitors but persisted in the presence of amiloride, which abolished the inhibitory effects of both nucleotides. Thus, apical nucleotides appear to evoke a transient stimulation of anion secretion and sustained inhibition of Na+ absorption. 5 Basolateral isoprenaline (10 microM) elicited a rise in ISC but subsequent addition of apical ATP reversed this effect. Conversely, isoprenaline restored ISC to its basal level following stimulation with ATP. Apical P2Y2 receptors and basolateral beta-adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.